Osteocytes are osteoblast-derived cells that are progressively entombed in mineralized matrix where they function to regulate skeletal homeostasis; their importance is highlighted both by their relative abundance and by their exceptionally long lifespan. Surprisingly, although osteocytes are known to manifest unique gene expression patterns relative to their osteoblast precursors and, as a consequence, display unique form and function, little is known of the molecular mechanisms that orchestrate their transition from the osteoblast or of the epigenomic and regulomic determinants that are responsible. In addition, although Wnt signaling is known to be a major but differential regulator of osteoblast and osteocyte activities, the majority of the gene targets that comprise the cell-specific activities of this pathway and the mechanisms through which TCF/LEF/b-catenin function to modulate these targets in both cell types remain unknown. Accordingly, the objectives of the first two aims are as follows. Aim 1: Identify the underlying epigenomic and regulomic mechanisms and associated molecular determinants that are responsible on a genome-wide scale for the osteoblast to osteocyte transition both in vitro and ex vivo. Aim 2: Examine and contrast the impact of the Wnt signaling pathway on osteoblast- and mature osteocyte-specific gene expression patterns and assess the molecular mechanisms through which these gene subsets are modulated directly by b-catenin and impacted by PTH and 1,25(OH)2D3 both in vitro and ex vivo. Our preliminary data and that of others have shown that a number of genes are upregulated during the osteocyte transition, including Sost, Fgf23, Tnfsf11, and Enpp1/3. The Sost gene, which encodes the Wnt pathway antagonist sclerostin (SCL), is of primary importance, however, due to its central biological actions on bone formation, its impact in disease, and its relevance as a potential therapeutic target. Progress has been made in understanding Sost regulation, prompted in part by the discovery that deletion of a downstream Sost enhancer alters Sost expression and is responsible for Van Buchem disease. Our preliminary data, however, suggest significant additional complexity, thus supporting the final objective of this proposal. Aim 3: Assess the molecular mechanisms that underlie both basal and regulated osteocyte expression of Sost at both epigenomic and regulomic levels in both in vitro and in vivo models. Osteocytes play unique roles in the skeleton and act in endocrine fashion to elaborate both local paracrine factors such as sclerostin and systemically active hormones such as FGF23. Pathologic consequences arising from aberrant osteocyte function can be catastrophic. Detailed basic insights arising from the proposed studies are likely to provide new routes of therapeutic intervention for a multiplicity of diseases that affect the skeleton.